Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobinhaptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae.
Monolayers of cytochrome P450scc and its complex with adrenodoxin
were formed by Langmuir techniques
and covalently immobilized on the solid substrates. The
orientation of hemeprotein molecules was studied
using polyclonal antibodies specific to the intact cytochrome P450scc
molecule and its tryptic fragments
F1 and F2, representing N- and C-terminal parts of the hemeprotein
molecule. Specific interactions of
the Langmuir films of cytochrome P450scc with adrenodoxin were
investigated, and the position of the
ferredoxin binding site at the hemeprotein molecule was identified.
It was shown that the molecular
orientation of the P450scc−AD (adrenodoxin) complex at the
water−air interface is dependent on the
surface density of the monolayer. The P450scc molecules do not
denature upon spreading on the water
surface. The formed monolayer can be transferred from the
air−water interface to the surface modified
with siloxane polymer and covalently immobilized without damage to the
structure. A model considering
the mode of orientation of cytochrome P450scc molecules on the
air−water interface in dependence on the
surface pressure is discussed.
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